The present invention relates to synthetic reagents or substrates which are used for the quantitative determination of proteolytic enzymes. More particlarly, the invention relates to a synthetic chromogenic or fluorescent tripeptidic substrate which is useful as a reagent for the quantitative determination of proteolytic enzymes of class E.C. 3.4.4., which split peptide chains on the carboxyl side of arginine as well as lysine, except thrombin and thrombin-like enzymes, in human and mammal body fluids as well as in vegetable and animal cell extracts and in glandular venoms of cold-blooded animals such as snakes.
The quantitative determination of kallikrein, a proteolytic enzyme which occurs in human and mammal body fluids (e.g. blood plasma) and organs (e.g. pancreas) and which has a vasodilatatory action, is carried out, on the one hand, by biological methods and, on the other hand, by enzymatic methods. The biological methods consist in measuring the changes caused by kallikrein in the carotis pressure of narcotized dogs, or in determining kallikrein on isolated guinea-pig intestine in tyrode solution by measuring the quantity of kinine released from kininase-free kininogen solutions (cf. E.K. FREY et al., "Das Kallikrein-Kinin-System und seine Inhibitoren", Ferdinand Enke Verlag, Stuttgart, 1968, p. 11 and 12). These biological methods have a major disadvantage because they require the cumbersome and expensive use of test animals. In order to overcome this difficulty, the so-called enzymatic determination methods were developed. These methods make use of the esterolytic action exerted on certain ester substrates by kallikrein. Substrates of this type include e.g. N.sup..alpha.-benzoyl-L-arginine ethyl ester which is split by kallikrein into N.sup..alpha.-L-arginine and ethyl alcohol. The rate of splitting can be measured e.g. by spectrophotometric determination of the increasing extinction at the wave length 253 nm (nanometer). Among other ester substrates which are proteolytically split by kallikrein and which are useful for kallikrein determinations the following can be mentioned: N.sup..alpha.-benzoyl-L-arginine methyl ester, N.sup..alpha.-toluenesulfonyl-L-arginine methyl ester and N.sup..alpha.-acetyl-L-tyrosine ethyl ester (cf. E.K. FREY et al., ibid., pages 12-14; and G. L. HABERLAND et al., "Kininogenases (Kallikrein)", 1st Symposium on Physiological Properties and Pharmacological Rational, F. K. Schattauer Verlag, Stuttgart-New York, 1973, p. 43 and 44). The enzymatic determination methods have the advantage of not requiring test animals, but have another serious disadvantage. The kallikrein solutions used for carrying out these methods must be optically clear and their concentration must be adjusted so that no substantial autoabsorption interferes at the wave length 253 nm. A further serious disadvantage of these enzymatic methods resides in the fact that the solution of the ester substrate (e.g. N.sup..alpha.-benzoyl-L-arginine ethyl ester) used for the determination of kallikrein have a high auto-absorption (the extinction of a 5 .times. 10.sup.-.sup.4 M solution in a cell of 1 cm at 253 nm is 2.1). At higher concentrations most UV spectrometers no longer show extinctions which are proportional to the concentrations used (cf. G. L. HABERLAND et al., ibid., p. 4).
A so-called amide substrate which is split amidolytically by certain enzymes of class E.C. 3.4.4. (in enzyme nomenclature the abbreviation "E.C." means "Enzyme Committee" of the "International Union of Biochemistry"), specifically thrombin and thrombin-like enzymes, is disclosed in German patent application DOS No. 2,322,116. This substrate comprises a base tripeptide chain of formula H-Phe-Val-Arg-OH which has its N-terminal amino acid blocked by a acyl group. The C-terminal amino acid carries as a substituent a chromogenic or fluorescent group which is split off by the proteolytic action of the said enzymes and yields a split product the quantity of which can be measured photometrically. This amide substrate is specifically adapted for the quantitative determination of thrombin and thrombin-like enzymes, but does not work at all in the determination of kallikrein, prekallikrein and similar enzymes.
The problem which had to be solved by the invention consisted in developing a new synthetic amide substrate which would be easily and rapidly split by kallikrein, more particularly plasma kallikrein, to yield split products which could easily be determined by spectrophotometric methods.
The solution of the said problem resulted from the following considerations: It was known that in humans suffering from or subjected to certain shocks or stresses the biologically highly active, pain-generating bradykinin is released from the naturally occurring substrate kininogen (molecular weight about 50,000) under the hydrolytic action of kallikrein. As a working hypothesis it was speculatively assumed that, by using a synthetic building unit corresponding to a C-terminal building unit of bradykinin and by attaching to the said synthetic building unit a chromogenic or fluorescent group, it might be possible to obtain a synthetic amide substrate which would have the same or almost the same susceptibility towards plasma kallikrein as the natural substrate and would yield by amidolysis a chromogenic or fluorescent split product having a high molecular extinction coefficient measurable at high wave lengths so as to minimize the influence of biological fluids on the measurement.
On the grounds of the above mentioned considerations a synthetic amide substrate meeting the defined requirements was developed.